Pharmacophore for trail induction

ABSTRACT

There are disclosed imidazolinopyrimidinone compounds that have activity to induce TRAIL gene expression in macrophages. There is further disclosed a method for treating various cancers comprising administering effective amounts of an imidazolinopyrimidinone having the structure of Formula I herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/359,633, filed on Mar. 20, 2019, which is a continuation of U.S.patent application Ser. No. 15/126,192, filed on Sep. 14, 2016, which isa U.S. national stage application filed under 35 U.S.C. § 371 fromInternational Application Serial No. PCT/US2015/023362, which filed onMar. 30, 2015, and published as WO 2015/153468 on Oct. 8, 2015, whichclaims the benefit of priority to U.S. Provisional Application Ser. No.61/972,689, filed Mar. 31, 2014, which applications and publication areincorporated by reference as if reproduced herein and made a part hereofin their entirety, and the benefit of priority of each of which isclaimed herein.

STATEMENT OF GOVERNMENT SUPPORT

The present invention was made with government support underHHSN27200700038C, AI077644, AI079436, and AI094348, awarded by theNational Institutes of Health. The U.S. government has certain rights inthe invention.

BACKGROUND

Cancer immunosurveillance relies on various effector functions of theimmune system that can modify both induced and spontaneouscarcinogenesis. TRAIL is an immunosurveillence cytokine criticallyinvolved in this process due to its ability to selectively induceapoptosis in cancer cells over normal cells (S. R. Wiley, K. Schooley,P. J. Smolak, W. S. Din, C. P. Huang, J. K. Nicholl, G. R. Sutherland,T. D. Smith, C. Rauch, C. A. Smith, Immunity 1995, 3, 673-682; A.Ashkenazi, V. M. Dixit, Science 1998; H. Walczak, R. E. Miller, K.Ariail, B. Gliniak, T. S. Griffith, M. Kubin, W. Chin, J. Jones, A.Woodward, T. Le, et al., Nat. Med. 1999, 5, 157-163; and A. Ashkenazi,R. C. Pai, S. Fong, S. Leung, D. A. Lawrence, S. A. Marsters, C.Blackie, L. Chang, A. E. McMurtrey, A. Hebert, et al., J. Clin. Invest.1999, 104, 155-162). The TRAIL gene is expressed in a variety of tissuesand cells (S. R. Wiley, K. Schooley, P. J. Smolak, W. S. Din, C. P.Huang, J. K. Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A.Smith, Immunity 1995, 3, 673-682); including dendritic cells, naturalkiller (NK) cells, and monocytes/macrophages (M. J. Smyth, K. Takeda, Y.Hayakawa, J. J. Peschon, M. R. M. van den Brink, H. Yagita, Immunity2003, 18, 1-6.). Its gene expression is under control of severaltranscriptional regulators, such as transcription factors NF-κB and p53(K. Kuribayashi, G. Krigsfeld, W. Wang, J. Xu, P. A. Mayes, D. T.Dicker, G. S. Wu, W. S. El-Deiry, Cancer Biol. Ther. 2008, 7,2034-2038.). Reduction of TRAIL expression by neutralizing antibodiesand ablation of TRAIL expression in mice lacking the TRAIL gene resultsin the development of carcinogen-induced fibrosarcomas, sarcomas, andlymphomas; especially in p53-deficient mice (E. Cretney, K. Takeda, H.Yagita, M. Glaccum, J. J. Peschon, M. J. Smyth, J. Immunol. 2002; and K.Takeda, M. J. Smyth, E. Cretney, Y. Hayakawa, N. Kayagaki, H. Yagita, K.Okumura, J. Exp. Med. 2002, 195, 161-169). These data are alsoconsistent with observations that change in TRAIL expression in immunecells is associated with TRAIL resistance in cancer cells (N. S. M.Azahri, M. M. Kavurma, Cell. Mol. Life Sci. 2013, 70, 3617-3629). Thus,effectors of TRAIL production in immune cells are of clinical relevance(M. J. Smyth, K. Takeda, Y. Hayakawa, J. J. Peschon, M. R. M. van denBrink, H. Yagita, Immunity 2003, 18, 1-6.) and could also be used as ameans to achieve a model system for studying the compleximmunosurveillance signaling system

SUMMARY

The invention is directed, in various embodiments, to a compound andpharmaceutical composition comprising an effective amount of a compoundcapable of inducing expression of TRAIL gene in cells capable ofexpressing the TRAIL gene to produce the cytokine TRAIL. TRAIL (acytokine) can selectively induce apoptosis in cancer cells over normalcells. Therefore, the present disclosure provides a compound andpharmaceutical that is effective for treating various cancers. Withoutbeing bound by theory, the disclosed compound and pharmaceuticalcomposition induces expression of TRAIL.

In various embodiments, the invention is directed to a compound offormula (I)

wherein

Cyc is a single 5- to 8-membered heterocyclyl ring comprising at leastone nitrogen atom, with a group of formula Ar¹—CR₂-being bonded to thenitrogen atom;

Ar¹ and Ar² are each independently selected aryl groups which areindependently substituted with 0, 1, or 2 J groups;

each independently selected R is H or is (C1-C6)alkyl;

J is (C1-C6)alkyl, (C3-C9)cycloalkyl, (C3-C9)cycloalkyl(C1-C6)alkyl, orhalo;

or a pharmaceutically acceptable salt thereof.

The present disclosure provides a pharmaceutical composition comprisinga compound selected from the group consisting of

In various embodiments, the compound used to induce TRAIL is compound 2

or a pharmaceutically acceptable salt thereof. The IUPAC name forcompound 2 is7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-one.

The present disclosure provides a method for treating various cancers,comprising administering to a patient an effective amount of a compoundof formula (I), such as compound 2. The method for treating a broadspectrum of mammalian cancers, wherein the broad spectrum of mammaliancancers to be treated is selected from the group consisting of ovarian,colon, breast, liver, pancreas, gastro-intestinal, head- and neck,cervix, prostate, lung cancers, melanomas, glioblastomas, myelomas,neuroblastic-derived CNS tumors, monocytic leukemias, B-cell derivedleukemias, T-cell derived leukemias, B-cell derived lymphomas, T-cellderived lymphomas, and mast cell derived tumors, and combinationsthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a) Induction of TRAIL mRNA in RAW cells treated for 48 hwith indicated dose of linear isomer (1) or angular isomer 2; b)Induction of TRAIL mRNA induction to 5 μM compound 1 and compound 2 forindicated times. c) Dose-dependent response to compound 2a and compound2b. d) Dose-dependent response to angular (2) and compound 9. Compound2a is a sample obtained from the NCI repository, compound 2b is acompound synthesized herein; both were shown to be a compound ofstructure 2.

FIG. 2 shows a comparison of the imidazolinopyrimidanone structures ofinactive compound 1 and active compound 2 with respect to TRAILexpression. The structures of each were confirmed by X-raycrystallographic analysis.

FIG. 3 shows the structure of constitutional isomer, compound 9.

FIG. 4 shows comparative structures of compound 1 and compound 2.

FIG. 5 shows the X-ray crystal structure obtained for compound 2.

FIG. 6 shows the X-ray crystal structure obtained for compound 9.

FIG. 7 shows a cell viability assay comparing the activity of a 20 mMconcentration of various compounds including Compound 2 (HIPPO) andcompounds A through R herein.

DETAILED DESCRIPTION

The present disclosure provides a compound of formula (I)

wherein

Cyc is a 5- to 8-membered monocyclic heterocyclyl ring comprising onenitrogen atom, with a group of formula Ar¹—CR₂-being bonded to the ringnitrogen atom;

Ar¹ and Ar² are each independently aryl groups which are substitutedwith 0, 1, or 2 J groups;

R is independently H or (C1-C6)alkyl;

J is independently (C1-C6)alkyl, (C3-C9)cycloalkyl,(C3-C9)cycloalkyl(C1-C6)alkyl, halo, or (C1-C6)haloalkyl;

or a pharmaceutically acceptable salt thereof.

Preferably, the compound of formula (I) is a compound within thesubgenus formula (IA)

or a pharmaceutically acceptable salt thereof.

More specifically, the compound of formula (IA) is a compound whereinAr¹ and Ar² are each a phenyl group substituted with 0, 1, or 2 Jgroups; and,

R at each occurrence is independently H or (C1-C6)alkyl;

or a pharmaceutically acceptable salt thereof.

Preferably, the compound of formula (I) is compound 2

or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides a compound of formula (I)that is not compound 2.

The present disclosure further provides a method for treating variouscancers, comprising administering an effective amount of a compound offormula (I)

wherein

Cyc is a 5- to 8-membered monocyclic heterocyclyl ring comprising onenitrogen atom, with a group of formula Ar¹—CR₂-being bonded to thenitrogen atom;

Ar¹ and Ar² are aryl groups which are substituted with 0, 1, or 2 Jgroups;

R is independently H or (C1-C6)alkyl;

J is independently (C1-C6)alkyl, (C3-C9)cycloalkyl,(C3-C9)cycloalkyl(C1-C6)alkyl, halo, or (C1-C6)haloalkyl;

or a pharmaceutically acceptable salt thereof.

Preferably, the compound is a compound selected from the subgenus offormula (IA)

or a pharmaceutically acceptable salt thereof.

More preferably, in the compound of formula (IA), Ar¹ and Ar² is aphenyl group substituted with 0, 1, or 2 J groups.

Most preferably the compound of formula (I) is compound 2

In various embodiments, the invention provides a method for treatingvarious cancers with a compound of formula (I) wherein the compound offormula (I) is not compound 2.

The method can be used for treating a broad spectrum of mammaliancancers, wherein the broad spectrum of mammalian cancers to be treatedis selected from the group consisting of ovarian, colon, breast, liver,pancreas, gastro-intestinal, head- and neck, cervix, prostate, lungcancers, melanomas, glioblastomas, myelomas, neuroblastic-derived CNStumors, monocytic leukemias, B-cell derived leukemias, T-cell derivedleukemias, B-cell derived lymphomas, T-cell derived lymphomas, and mastcell derived tumors, and combinations thereof.

Another imidazolinopyrimidinone, (called compound 1 herein) in disclosedin United States patent application 20120276088 published 1 Nov. 2012.This patent application discloses linear compound 1 which is used forcomparison purposes herein. We synthesized compound 1 in four steps from4-chloronicotinic acid (Scheme 1).

Synthesis of Compound 1

(a) SOCl₂, 90° C., 1 h, then 2-methylthioimidazoline hydroiodide, Et₃N,CH₂Cl₂, 0° C. to rt, 19 h, 96%; (b) 2-methylbenzylamine, K₃PO₄,N,N-dimethylacetamide, reflux, 1 h, 79%; (c) 45 psi H₂(g), PtO₂,MeOH/TFA, rt, 5 h, 80%; (d) benzaldehyde, Na(OAc)₃BH, AcOH, CH₂Cl₂, rt,4 h, 87%.

Briefly, acylation of an activated carboxylic acid, followed by a doubledisplacement reaction, and subsequent hydrogenation and reductiveamination afforded compound 1 in 52% overall yield. This structure ofcompound 1 was confirmed by mass spectrometry, nuclear magneticresonance (NMR) spectroscopic, and X-ray crystallographic analyses (seeExamples section).

The biological activity of compound 1 was measured by RT-PCR analysis ofTRAIL mRNA expression in the murine macrophage cell line RAW 264.7. Nochange in TRAIL mRNA expression over controls was observed, even atdoses as high as 10 μM (FIG. 1a ) or with prolonged exposure (FIG. 1b ).As shown in FIG. 1, compound 2a, (obtained from the NCI), exhibits thedesired TRAIL bioactivity, as did synthesized compound 2b, butsynthesized compound 1 does not. Therefore, there is a need in the artto create a biologically active imidazolinopyrimidinone, which is themore angular compound of formula (I), and in particular compound 2.

Compound 2 was prepared in three steps in 82% yield (Scheme 2). Asynthetic product, termed herein compound 2b, was obtained, and itsstructure confirmed as 2.

Synthesis of Compound 2

(a) methyl chloroformate, Et₃N, CH₂Cl₂, 0° C. to rt, 44 h, 97%; (b)2-methylbenzylamine, MeOH, AcOH, reflux, 45 h, 87%; (c) NaOMe, MeOH,reflux, 18 h, 97%.

A mixture of guanidine 7 and 1-benzyl-4-oxopiperidine-3-carboxylatehydrochloride (8) in refluxing methanol and sodium methoxide afforded 2balmost exclusively; a trace amount of 1 was detected by ¹H NMR followingwork-up of this reaction, but was removed by subsequent purification. Werationalize this result by considering that the imidazolinyl nitrogensof 7 possess both statistical and steric advantages over the benzylicnitrogen of 7. Initial attack by nitrogen at the ketone carbonyl of 8affords an aminocarbinol intermediate, which suffers intramolecularcyclocondensation to provide synthetic sample 2b. Its structure 2 wasconfirmed by mass spectrometry and NMR spectroscopy.

Compound 2, obtained as synthetic sample 2b was able to induce TRAILmRNA expression, as did repository compound 2a (FIG. 1c ).

Therefore, angular compound 2 (shown by the inventors herein to be theactive TRAIL induction factor) has the structure

Compound 1 (does not seem to be active) has the structure

and the isomeric linear compound to have the structure 9

Of these three compounds, only compound 2 exhibits the desired TRAILbioactivity.

X-ray crystal structures, taken as described in the Examples section,are provided in the Figures.

These findings provide a structure-activity relationship wherein theangular fusion of the tricyclic core is a necessity of the pharmacophorefor TRAIL induction in macrophages.

Our three-step synthesis of compound 2 began with the preparation ofcarbamate 6 (T. Smejkal, D. Gribkov, J. Geier, M. Keller, B. Breit,Chemistry 2010, 16, 2470-2478) and its conversion to guanidine 7 (W. K.Fang, P. X. Nguyen, K. Chow, T. M. Heidelbaugh, D. G. Gomez, M. E.Garst, S. C. Sinha, Allergan Inc., USA, 2011). If the 1,1-diamine isunsymmetrical, an isomeric mixture of products is possible (see: J. V.Greenhill, M. J. Ismail, P. N. Edwards, P. J. Taylor, J Chem Soc Perk T2 1985, 1255-1264; C. Romano, E. Delacuesta, C. Avendano, F. Florencio,J. Sainzaparicio, Tetrahedron 1988, 44, 7185-7192; F. Esser, K. H. Pook,A. Carpy, Synthesis-Stuttgart 1990, 72-78). A mixture of guanidine 7 and1-benzyl-4-oxopiperidine-3-carboxylate hydrochloride in refluxingmethanol (with the aid of NaOMe) afforded compound 2 almost exclusively;a trace amount of compound 1 was detected by ¹H NMR following work-up ofthis reaction. We rationalize this result by considering that theimidazolinyl nitrogens of 7 possess both statistical and stericadvantages over the benzylic nitrogen of 7. Initial attack by nitrogenat the ketone carbonyl affords an aminocarbinol intermediate, whichsuffers intramolecular cyclocondensation to provide 2.

The K₂CO₃-mediated reaction of a 3-keto ester with a 2-amino-2-oxazoline(a type of unsymmetrical 1,1-diamine) affords a mixture of linear andangular products (I. Forfar, C. Jarry, M. Laguerre, J. M. Leger, I.Pianet, Tetrahedron 1999, 55, 12819-12828). The authors accumulatedempirical and theoretical evidence to support the notion that “theendocyclic nitrogen atom is the most nucleophilic and attacks the mostelectrophilic carbon of the biselectrophile. A ring closure between theexocyclic nitrogen atom and the second electrophilic center concludesthe bicyclic heterocycle synthesis.” This is consistent with our ownobservations in the synthesis of 7 via a similar strategy.

To reiterate the salient feature of the present synthesis, by usingsodium methoxide in refluxing methanol (M. F. Koehler, P. Bergeron, E.Blackwood, K. K. Bowman, Y. H. Chen, G. Deshmukh, X. Ding, J. Epler, K.Lau, L. Lee, L. Liu, C. Ly, S. Malek, J. Nonomiya, J. Oeh, D. F.Ortwine, D. Sampath, S. Sideris, L. Trinh, T. Truong, J. Wu, Z. Pei, J.P. Lyssikatos, J. Med. Chem. 2012, 55, 10958-10971), compound 2 isproduced nearly exclusively. If the condensation is performed in thepresence of base and/or at higher temperature, then sufficient means areavailable for statistically and sterically more likely aminocarbinolintermediate to suffer rapid intramolecular cyclocondensation leading tocompound 2.

In addition, related compounds A through R were synthesized. Thecharacteristics of compounds A through R are provided in Table 1 below:

TABLE 1 Compound Label Structure Chemical Info A

Chemical Formula: C23H24N4O Molecular Weight: 372.47 Log P: 2.6 B

Chemical Formula: C24H26N4O Molecular Weight: 386.50 Log P: 3.09 C

Chemical Formula: C23H23BrN4O Molecular Weight: 451.37 Log P: 3.43 D

Chemical Formula: C23H23ClN4O Molecular Weight: 406.91 Log P: 3.16 E

Chemical Formula: C17H20N4O Molecular Weight: 296.37 Log P: 0.87 F

Chemical Formula: C24H26N4O2 Molecular Weight: 402.50 Log P: 2.47 G

Chemical Formula: C17H20N4O Molecular Weight: 296.37 Log P: 0.87 H

Chemical Formula: C18H22N4O Molecular Weight: 310.40 Log P: 1.35 I

Chemical Formula: C17H19BrN4O Molecular Weight: 375.27 Log F: 1.7 J

Chemical Formula: C17H19ClN4O Molecular Weight: 330.82 Log P: 1.42 K

Chemical Formula: C11H16N4O Molecular Weight: 220.28 Log P: −0.87 L

Chemical Formula: C18H22N4O2 Molecular Weight: 326.40 Log P: 0.74 M

Chemical Formula: C17H19N3O Molecular Weight: 281.36 Log P: 2.29 N

Chemical Formula: C18H21N3O Molecular Weight: 295.39 Log P: 2.78 O

Chemical Formula: C17H18BrN3O Molecular Weight: 360.26 Log P: 3.12 P

Chemical Formula: C17H18ClN3O Molecular Weight: 315.80 Log P: 2.85 Q

Chemical Formula: C11H15N3O Molecular Weight: 205.26 Log P: 0.56 R

Chemical Formula: C18H21N3O2 Molecular Weight: 311.38 Log P: 2.17

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

The term “about” as used herein, when referring to a numerical value orrange, allows for a degree of variability in the value or range, forexample, within 10%, or within 5% of a stated value or of a stated limitof a range.

All percent compositions are given as weight-percentages, unlessotherwise stated.

The term “disease” or “disorder” or “malcondition” are usedinterchangeably, and are used to refer to diseases or conditions whereinTRAIL, such as inducing expression of the TRAIL gene in a cell, plays arole in the biochemical mechanisms involved in the disease ormalcondition or symptom(s) thereof such that a therapeuticallybeneficial effect can be achieved with an effective amount orconcentration of a synthetic ligand of the invention adequate to induceexpression of TRAIL and induce apoptosis, e.g., selectively in cancercells. For example, the cancers to be treated by the compounds of thepresent disclosure include a broad spectrum of mammalian cancers,wherein the broad spectrum of mammalian cancers to be treated isselected from the group consisting of ovarian, colon, breast, lungcancers, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias,B-cell derived leukemias, T-cell derived leukemias, B-cell derivedlymphomas, T-cell derived lymphomas, and mast cell derived tumors, andcombinations thereof.

The expression “effective amount”, when used to describe therapy to anindividual suffering from a disorder, refers to the quantity orconcentration of a compound of the invention that is effective to induceexpression of TRAIL in the individual's tissues.

The terms “halo” or “halogen” or “halide” by themselves or as part ofanother substituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.

A “salt” as is well known in the art includes an organic compound suchas a carboxylic acid, a sulfonic acid, or an amine, in ionic form, incombination with a counterion. For example, acids in their anionic formcan form salts with cations such as metal cations, for example sodium,potassium, and the like; with ammonium salts such as NH₄ ⁺or the cationsof various amines, including tetraalkyl ammonium salts such astetramethylammonium, or other cations such as trimethylsulfonium, andthe like. A “pharmaceutically acceptable” or “pharmacologicallyacceptable” salt is a salt formed from an ion that has been approved forhuman consumption and is generally non-toxic, such as a chloride salt ora sodium salt. A “zwitterion” is an internal salt such as can be formedin a molecule that has at least two ionizable groups, one forming ananion and the other a cation, which serve to balance each other. Forexample, amino acids such as glycine can exist in a zwitterionic form. A“zwitterion” is a salt within the meaning herein. The compounds of thepresent invention may take the form of salts. The term “salts” embracesaddition salts of free acids or free bases which are compounds of theinvention. Salts can be “pharmaceutically-acceptable salts.” The term“pharmaceutically-acceptable salt” refers to salts which possesstoxicity profiles within a range that affords utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present invention, such as for example utility inprocess of synthesis, purification or formulation of compounds of theinvention. “Pharmaceutically or pharmacologically acceptable” includemolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. For human administration, preparations shouldmeet sterility, pyrogenicity, and general safety and purity standards asrequired by FDA Office of Biologics standards.

Examples General Procedures

All reactions were carried out under an argon atmosphere with drysolvents using anhydrous conditions unless otherwise stated. Chemicalswere purchased from Acros Organics, Oakwood Products, and Sigma-Aldrich.They were used as received unless otherwise noted. Dry dichloromethane(CH₂Cl₂) was obtained via distillation over calcium hydride (CaH₂). Drymethanol (MeOH) was obtained via distillation over magnesium turnings.Reagents were purchased at the highest commercial quality and usedwithout further purification, unless otherwise stated. Yields refer tochromatographically and spectroscopically (¹H NMR) homogeneousmaterials, unless otherwise stated. Reactions were monitored by thinlayer chromatography (TLC) carried out on 0.25 mm E. Merck silica gelplates (60F-254) using UV light as the visualizing agent, or basicaqueous potassium permanganate (KMnO₄), and heat as developing agent. E.Merck silica gel (60, particle size 0.040-0.063 mm) was used for flashcolumn chromatography. Preparative thin layer chromatography (PTLC)separations were carried out on 0.50 mm E. Merck silica gel plates(60F-254). Concentration of organic solvents was performed on a rotaryevaporator under reduced pressure followed by further evacuation using adual stage mechanical pump. NMR spectra were recorded on Bruker DRX-600,DRX-500, and AMX-400 instruments and calibrated using residualundeuterated solvent as an internal reference (CHCl₃ @ δ 7.26 ppm ¹HNMR, δ 77.16 ppm ¹³C NMR; CD₃OD @ δ 4.87 ppm ¹H NMR, δ 49.00 ppm 13CNMR). The following abbreviations (or combinations thereof) were used toexplain ¹H NMR multiplicities: s=singlet, d=doublet, t=triplet,m=multiplet, br=broad. High-resolution mass spectra (HRMS) were recordedon Agilent LC/MSD TOF mass spectrometer by electrospray ionizationtime-of-flight reflectron experiments. IR spectra were recorded oneither a PerkinElmer Spectrum 100 FTIR spectrometer with ATR accessoryor a Jasco 480 Plus FTIR spectrometer. Melting points were recorded on aFisher-Johns 12-144 melting point apparatus and are uncorrected.

Synthetic Procedures

(4-Chloropyridin-3-yl)(2-(methylthio)-4,5-dihydro-1H-imidazol-1-yl)methanone(3)

A mixture of 4-chloronicotinic acid (1.00 g, 6.35 mmol) and SOCl₂ (15mL) was stirred at 90° C. for 1 h. Removal of SOCl₂ by rotaryevaporation gave 4-chloronicotinic acid chloride hydrochloride as a paleyellow solid, which was placed under argon balloon, cooled to 0° C., anddissolved in CH₂Cl₂ (45 mL). A solution of 2-methylthio-2-imidazolinehydriodide (1.32 g, 5.40 mmol) and Et₃N (2.92 mL, 20.95 mmol) in CH₂Cl₂(75 mL) was added via cannula. The pale amber solution was stirred atroom temperature overnight. After 19 h, CH₂Cl₂ (150 mL) was added andthe resulting solution washed with saturated aqueous NaHCO₃ (2×100 mL)and brine (2×100 mL). The organic layer was dried (Na₂SO₄) andconcentrated in vacuo. Purification by silica gel chromatography (19:1CH₂Cl₂/MeOH) afforded 3 (1.32 g, 96%) as a pale yellow syrup.

R_(f)=0.19 (silica gel, 19:1 CH₂Cl₂/MeOH)

IR (neat) ν_(max) 1661, 1574, 1377, 1200, 903, 724 cm⁻¹

¹H NMR (600 MHz, CDCl₃) δ 8.56 (d, J=5.5 Hz, 1H), 8.54 (s, 1H), 7.37 (d,J=5.2 Hz, 1H), 4.15-3.65 (m, 2H), 3.93 (t, J=8.3 Hz, 2H), 2.37 (s, 3H)

¹³C NMR (150 MHz, CDCl₃) δ 162.1, 151.9, 148.6, 131.9, 124.7, 54.1,48.5, 15.6

HRMS (ESI-TOF) calcd. for C₁₀H₁₀ClN₃OSH⁺ [M+H⁺] 256.0306, found 256.0309

10-(2-Methylbenzyl)-2,3-dihydroimidazo[1,2-a]pyrido[4,3-d]pyrimidin-5(10H)-one(4)

A mixture of 3 (1.30 g, 5.08 mmol), 2-methylbenzylamine (1.89 mL, 15.25mmol), powdered K₃PO₄ (1.08 g, 5.08 mmol), and N,N-dimethylacetamide (10mL) was heated at reflux for 1 h. The resulting mixture was cooled andpartitioned between CH₂Cl₂ (30 mL) and H₂O (30 mL). The organic layerwas dried (Na₂SO₄) and concentrated in vacuo. Purification by silica gelchromatography (19:1 CH₂Cl₂/MeOH) and trituration with cold hexanesafforded 4 (1.17 g, 79%) as a white solid.

m.p. 182-188° C. (hexanes)

R_(f)=0.32 (silica gel, 19:1 CH₂Cl₂/MeOH)

IR (neat) ν_(max) 1674, 1634, 1591, 1455, 1400, 1284, 747 cm⁻¹

¹H NMR (500 MHz, CDCl₃) δ 9.15 (s, 1H), 8.45 (d, J=5.9 Hz, 1H), 7.23 (d,J=7.4 Hz, 1H), 7.19 (t, J=7.4 Hz, 1H), 7.11 (t, J=7.4 Hz, 1H), 6.84 (d,J=7.7 Hz, 1H), 6.55 (d, J=5.9 Hz, 1H), 5.21 (s, 2H), 4.20 (t, J=8.9 Hz,2H), 3.96 (t, J=8.9 Hz, 2H), 2.41 (s, 3H)

¹³C NMR (150 MHz, CDCl₃) δ 158.0, 154.6, 151.0, 150.2, 147.7, 135.0,131.6, 131.0, 127.8, 126.7, 124.2, 111.9, 107.9, 50.2, 46.7, 45.3, 19.2

HRMS (ESI-TOF) calcd. for C₁₇H₁₆N₄OH⁺ [M+H⁺] 293.1397, found 293.1397

10-(2-Methylbenzyl)-2,3,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[4,3-d]pyrimidin-5(10H)-one(5)

A mixture of 4 (300 mg, 1.03 mmol), PtO₂ (60 mg), MeOH (3 mL), and TFA(3 mL) was hydrogenated (45 psi) in a Parr shaker for 5 h. The mixturewas filtered through a Celite pad to remove catalyst, then concentratedin vacuo. The colorless syrup was dissolved in 1:1 EtOAc/H₂O (40 mL),made basic by addition of 2 M NaOH (10 mL), and layers were separated.The aqueous layer was extracted with EtOAc (40 mL). The combined organiclayers were washed with brine (20 mL), dried (Na₂SO₄), and concentratedin vacuo. Purification by silica gel chromatography (19:1:0.1CH₂Cl₂/MeOH/NH₄OH) afforded 5 (244 mg, 80%) as a white solid.

m.p. 170-174° C. (MeOH)

R_(f)=0.12 (silica gel, 19:1:0.1 CH₂Cl₂/MeOH/NH₄OH)

IR (neat) ν_(max) 3287, 1660, 1627, 1605, 1472, 1293, 919 cm⁻¹

¹H NMR (600 MHz, CDCl₃) δ 7.20-7.14 (m, 3H), 6.92-6.90 (m, 1H), 4.98 (s,2H), 4.05 (t, J=9.4 Hz, 2H), 3.82 (t, J=9.4 Hz, 2H), 3.68 (t, J=1.9 Hz,2H), 2.95 (t, J=5.8 Hz, 2H), 2.30 (s, 3H), 2.28-2.25 (m, 2H), 1.66 (brs, 1H)

¹³C NMR (150 MHz, CDCl₃) δ 160.0, 152.8, 147.2, 134.6, 133.8, 130.7,127.4, 126.8, 123.7, 106.6, 49.9, 46.0, 45.2, 42.7, 42.2, 25.5, 19.1

HRMS (ESI-TOF) calcd. for C₁₇H₂₀N₄OH⁺ [M+H⁺] 297.1710, found 297.1709

7-Benzyl-10-(2-methylbenzyl)-2,6,7,8,9,10-hexahydroimidazo[1,2-a]pyrido[4,3-d]pyrimidin-5(3H)-one(1)

A solution of 5 (230 mg, 0.78 mmol) and benzaldehyde (103 μL, 1.02 mmol)in CH₂Cl₂ (2.5 mL) was treated with AcOH (76 μL, 1.35 mmol) andNa(OAc)₃BH (267 mg, 1.26 mmol) at room temperature. The mixture wasstirred for 4 h, then diluted with CH₂Cl₂ (10 mL) and washed withsaturated aqueous NaHCO₃ (10 mL). The aqueous layer was extracted withCH₂Cl₂ (10 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated in vacuo. Purification by silica gel chromatography (19:1CH₂Cl₂/MeOH) afforded 1 (261 mg, 87%) as a white solid.

m.p. 166-168° C. (MeOH)

R_(f)=0.25 (silica gel, 19:1 CH₂Cl₂/MeOH)

IR (neat) ν_(max) 2866, 2358, 2339, 1616, 1456, 983 cm⁻¹

¹H NMR (600 MHz, CDCl₃) δ 7.37-7.28 (m, 4H), 7.26-7.14 (m, 4H),6.93-6.91 (m, 1H), 4.98 (s, 2H), 4.06 (t, J=9.4 Hz, 2H), 3.84 (t, J=9.4Hz, 2H), 3.64 (s, 2H), 3.38 (s, 2H), 2.54 (t, J=5.7 Hz, 2H), 2.37 (t,J=5.5 Hz, 2H), 2.29 (s, 3H)

¹³C NMR (150 MHz, CDCl₃) δ 159.8, 152.9, 147.1, 137.6, 134.6, 133.7,130.7, 129.2, 128.5, 127.5, 127.4, 126.8, 123.7, 105.7, 62.1, 49.9,49.6, 48.6, 46.4, 45.3, 26.1, 19.1

HRMS (ESI-TOF) calcd. for C₂₄H₂₆N₄OH⁺ [M+H⁺] 387.2179, found 387.2189

Methyl 2-(methylthio)-4,5-dihydro-1H-imidazole-1-carboxylate (6)

A solution of 2-methylthio-2-imidazoline hydriodide (12.21 g, 50 mmol)and Et₃N (16 mL, 115 mmol) in CH₂Cl₂ (50 mL) at 0° C. was treated withmethyl chloroformate (5.0 mL, 65 mmol) dropwise. The mixture was allowedto warm to room temperature and stirred overnight. After 44 h, themixture was diluted with EtOAc (200 mL), stirred, then filtered toremove insoluble salts. The salts were rinsed with EtOAc (50 mL). Thefiltrate was concentrated in vacuo, affording 6 (8.47 g, 97%) as a whitesolid.

R_(f)=0.33 (silica gel, 19:1 CH₂Cl₂/MeOH)

IR (neat) ν_(max) 1717, 1576, 1429, 1378, 1218, 1023, 758 cm⁻¹

¹H NMR (600 MHz, CDCl₃) δ 3.92-3.85 (m, 4H), 3.78 (s, 3H), 2.41 (s, 3H)

¹³C NMR (150 MHz, CDCl₃) δ 159.7, 152.5, 53.9, 53.2, 47.5, 15.2

HRMS (ESI-TOF) calcd. for C₆H₁₀N₂O₂SH⁺ [M+H⁺] 175.0536, found 175.0539

N-(2-Methylbenzyl)-4,5-dihydro-1H-imidazol-2-amine (7)

A solution of 6 (1.5 g, 8.61 mmol) and 2-methylbenzylamine (1.08 mL,8.74 mmol) in MeOH (48 mL) was treated with AcOH (4.8 mL). The solutionwas stirred at a gentle reflux. After 45 h, the solution was cooled toroom temperature and concentrated in vacuo. The residue was dissolved inCH₂Cl₂ (100 mL), washed with 1 M NaOH (55 mL), brine (55 mL), dried overNa₂SO₄, filtered, and concentrated in vacuo. Trituration with cold CH₃CNafforded 7 (1.42 g, 87%) as a white solid.

R_(f)=0.14 (silica gel, 9:1:0.1 CH₂Cl₂/MeOH/NH₄OH)

IR (neat) ν_(max) 2862, 2358, 1684, 1635, 1521, 1349, 1238 cm⁻¹

¹H NMR (600 MHz, CD₃OD) δ 7.25-7.15 (m, 4H), 4.34 (s, 2H), 3.61 (s, 4H),2.32 (s, 3H)

¹³C NMR (150 MHz, CD₃OD) δ 163.0, 161.5, 137.4, 136.7, 131.4, 128.8,128.5, 127.2, 46.2, 45.8, 18.9

HRMS (ESI-TOF) calcd. for C₁₁H₁₅N₃H⁺ [M+H⁺] 190.1339, found 190.1344.

7-Benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(4H)-one(2)

A mixture of methyl 1-benzyl-4-oxopiperidine-3-carboxylatehydrochloride, 8, (568 mg, 2.0 mmol) and 7 (795 mg, 4.2 mmol) wastreated with a solution of sodium methoxide in MeOH (0.5 M, 3.0 mL, 1.5mmol). The mixture was stirred at a gentle reflux overnight. After 18 h,the reaction was cooled to room temperature, diluted with CH₂Cl₂ (50mL), washed with brine (20 mL), dried over Na₂SO₄, filtered, andconcentrated in vacuo. Purification by silica gel chromatography (19:1CH₂Cl₂/MeOH) afforded 2 (753 mg, 97%) as a pale yellow solid.

m.p. 132-135° C. (MeOH)

R_(f)=0.25 (silica gel, 19:1 CH₂Cl₂/MeOH)

IR (neat) ν_(max) 2750, 2358, 1646, 1616, 1487, 1296, 738 cm⁻¹

¹H NMR (500 MHz, CDCl₃) δ 7.33 (m, 5H), 7.11 (m, 4H), 5.05 (s, 2H), 3.89(m, 4H), 3.67 (s, 2H), 3.32 (s, 2H), 2.68 (m, 2H), 2.51 (m, 2H), 2.40(s, 3H)

¹³C NMR (150 MHz, CDCl₃) δ 161.6, 153.4, 145.8, 137.7, 135.7, 134.4,130.4, 129.3, 128.6, 127.5, 127.0, 126.0, 125.4, 102.1, 62.5, 50.7,49.7, 48.3, 47.1, 43.3, 27.0, 19.4

HRMS (ESI-TOF) calcd. for C₂₄H₂₆N₄OH⁺ [M+H⁺] 387.2179, found 387.2166

TABLE 1 Comparison of 13C NMR chemical shifts for compounds 1, 2, and 9JWL JWL NCI MK (1) (2) (2) (9) 159.8 161.6 161.5 160.7 152.9 153.4 153.4160.4 147.1 145.8 145.8 154.5 137.6 137.7 137.6 138.4 134.6 135.7 135.7137.0 133.7 134.4 134.3 133.6 130.7 130.4 130.3 130.9 129.2 129.3 129.3129.3 128.5 128.6 128.6 129.0 127.5 127.5 127.5 128.5 127.4 127.0 126.9128.2 126.8 126.0 126.0 127.3 123.7 125.4 125.3 126.3 105.7 102.1 102.2109.2 62.1 62.5 62.4 62.7 49.9 50.7 50.5 50.1 49.6 49.7 49.6 49.6 48.648.3 48.3 46.9 46.4 47.1 47.1 44.5 45.3 43.3 43.3 40.6 26.1 27.0 26.932.4 19.1 19.4 19.4 19.3 Spectra were recorded at 150 MHz in CDCl3.

X-Ray Crystal Structures

The X-ray crystal structures of compounds 2 (as synthetic sample 2b) and9 were obtained. The parameters are given below, and the structuresobtained provided in FIGS. 5 and 6, respectively.

Compound 2 (Also Called HIPPO)

The single crystal X-ray diffraction studies were carried out on aBruker X8 APEX II Ultra CCD diffractometer equipped with Mo Kα radiation(λ=0.71073). A 0.18×0.16×0.08 mm clear colorless plate of 2 was mountedon a Cryoloop with Paratone oil. Data were collected in a nitrogen gasstream at 100 K using ω scans. Crystal-to-detector distance was 50 mmusing 5 s exposure time with a 1.0° scan width. Data collection was99.9% complete to 25.00° in 0. A total of 14019 reflections werecollected covering the indices, −11<=h<=10, −11<=k<=11, −19<=l<=18. 4833reflections were found to be symmetry independent, with a Rint of0.0391. Indexing and unit cell refinement indicated a primitive,triclinic lattice. The space group was found to be P-1. The data wereintegrated using the Bruker SAINT software program and scaled using theSADABS software program. Solution by direct methods (SHELXT) produced acomplete phasing model consistent with the proposed structure.

All non-hydrogen atoms were refined anisotropically by full-matrixleast-squares (SHELXL). All hydrogen atoms were placed using a ridingmodel. Their positions were constrained relative to their parent atomusing the appropriate HFIX command in SHELXL.

Crystallographic data are summarized below. Full metrical parameters areavailable from the CCDC under number 981022. See FIG. 5.

Crystal Data and Structure Refinement for Compound 2

Identification code Janda01 (2) Empirical formula C₂₄ H₂₆ N₄O Molecularformula C₂₄ H₂₆ N₄O Formula weight 386.49 Temperature 100 K Wavelength0.71073 Å Crystal system Triclinic Space group P-1 Unit cell dimensionsa = 8.1173(11) Å α = 85.638(3)° b = 8.4320(11) Å β = 85.045(3)° c =14.6360(19) Å γ = 83.059(3)° Volume 988.5(2) Å³ Z 2 Density (calculated)1.298 Mg/m³ Absorption coefficient 0.082 mm⁻¹ F(000) 412 Crystal size0.18 × 0.16 × 0.08 mm³ Crystal color, habit colorless plate Theta rangefor data collection 2.439 to 29.252° Index ranges −11 <= h <= 10, −11 <=k <= 11, −19 <= l <= 18 Reflections collected 14019 Independentreflections 4833 [R(int) = 0.0391] Completeness to theta = 99.9% 25.000°Absorption correction Semi-empirical from equivalents Max. and min.transmission 0.0976 and 0.0673 Refinement method Full-matrixleast-squares on F² Data/restraints/parameters 4833/0/263Goodness-of-fit on F2 1.027 Final R indices R1 = 0.0433, wR2 = 0.1082[I > 2sigma(I)] R indices (all data) R1 = 0.0697, wR2 = 0.1181Extinction coefficient n/a Largest diff. peak and hole 0.320 and −0.204e.Å⁻³

A colorless crystal of compound 9 was mounted on a Cryoloop withParatone oil and data was collected at 100 K on a Bruker APEX II CCDwith Mo K_(a) radiation (generated from a Mo rotating anode). Data wascorrected for absorption with SADABS and structure was solved by directmethods.

All non-hydrogen atoms were refined anisotropically by full-matrixleast-squares on F² and all hydrogen atoms were placed in calculatedpositions with appropriate riding parameters.

Highest peak 0.20 at 0.4224 0.6962 0.1821 [0.63 A from C9]Deepest hole −0.23 at 0.0912 0.4660 0.3644 [0.93 A from C17]Crystallographic parameters are summarized below. Full metricalparameters are available from the CCDC under number 981024. See FIG. 6.

Crystal Data and Structure Refinement for Compound 9

Identification code Janda03 (9) Empirical formula C₂₄ H₂₆ N₄ O Molecularformula C₂₄ H₂₆ N₄ O Formula weight 386.49 Temperature 100 K Wavelength0.71073 Å Crystal system Triclinic Space group P-1 Unit cell dimensionsa = 5.6439(18) Å α = 93.194(9)° b = 10.537(4) Å β = 91.021(6)° c =16.502(5) Å γ = 96.745(5)° Volume 972.8(6) Å³ Z 2 Density (calculated)1.319 Mg/m³ Absorption coefficient 0.083 mm⁻¹ F(000) 412 Crystal size0.22 × 0.02 × 0.02 mm³ Crystal color, habit colorless rod Theta rangefor data collection 1.95 to 26.34° Index ranges −6 <= h <= 6, −13 <= k<= 12, −20 <= l <= 18 Reflections collected 10564 Independentreflections 3904 [R(int) = 0.0507] Completeness to theta = 25.00° 99.9%Absorption correction multi-scan/SADABS Max. and min. transmission0.9983 and 0.9820 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 3904/0/263 Goodness-of-fit on F² 1.003 FinalR indices [I > 2sigma(I)] R1 = 0.0430, wR2 = 0.0942 R indices (all data)R1 = 0.0719, wR2 = 0.1068 Largest diff. peak and hole 0.201 and −0.229e.Å⁻³

Biological Methods Cell Culture Methods:

RAW 264.7 cells (ATCC TIB-71) were maintained in growth medium ofDulbecco's Modified Eagle's Medium (DMEM with 4.5 g/L glucose andpyruvate, Gibco BRL, Invitrogen Corp., USA) supplemented withL-glutamine, penicillin/streptomycin, non-essential amino acids (100×stocks, Invitrogen Corp.), 10 mM HEPES, pH 7.4 (1 M stock, Invitrogen),and 10% Fetal Bovine Serum (FBS, Hyclone); (V. V. Kravchenko, R. J.Ulevitch, G. F. Kaufmann, Methods Mol. Biol. 2011, 692, 133-145).

RNA Rt-PCR Experiments:

Cells were plated in 6-well plates (Corning Costar 3506) diluted 1:5 in3 mL growth medium, media was changed after cells had adhered. After 12h incubation, cells were treated with described concentration ofcompound in DMSO, and incubated in the presence of that compound orvehicle for the described amount of time. At this time, media wasremoved and cells were treated with TRIzol reagent (Life Technologies),and RNA extracted via included protocol. RNA concentration determinedusing a Hitachi U-2000 UV-Vis Spectrophotometer and samples diluted to12 μg/5 μL in H2O. This solution was diluted 1:5 in H₂O and 1 μL of thissolution was mixed with 50 μL of RT-PCR reaction mixture (Qiagen OnestepRT-PCR kit) and TRAIL primers.

Mouse:

mTRAIL-F: 5′-GACACCATTTCTACAGTTCCAG-3′ (SEQ ID NO. 1),mTRAIL-R: 5′-CGGATAGCTGGTGTACTTGTAG-3′ 3′ (SEQ ID NO. 2).

Human:

hTrail-F2: 5′-ACAGACCTGCGTGCTGATCGTG-3′ 3′ (SEQ ID NO. 3) (exon 1)hTrail-R2: 5′-ACGAGCTGACGGAGTTGCCAC-3′ 3′ (SEQ ID NO. 4) (exon 2).

RT-PCR was run on an Applied Biosystems Gene Amp 9700 PCR system. RT-PCRproducts were analyzed on 5.5% acrylamide gel in TAE buffer (T.Maniatis, E. F. Fritsch, J. Sambrook, Molecular Cloning: a LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989).

RAW 264.7 cells were plated at 500 cell/well in Costar 96-well plates(Corning Inc, NY) in phenol-free Dulbecco's Modified Eagle's Medium(DMEM with 4.5 g/L glucose, Gibco BRL, Invitrogen Corporation, USA)supplemented with 10% fetal bovine serum (Gibco BRL, Invitrogen Corp.,USA), L-glutamine, pyruvate, penicillin/streptomycin, and nonessentialamino acids (100× stocks from Invitrogen). After 4 hours, cells werethen treated in triplicate with vehicle, lysis buffer, 20 μM of compound2 (HIPPO), or 20 μM of 18 derivative compounds (A through R) as listedin Table 1, above. After 48 hours, cell viability was assessed bycolorimetric XTT formazan assay (Cell Signaling Tech.) according tomanufacturer protocol. Relative absorbance was normalized to the vehicletreated cells (negative control) and lysis buffer treated cells(positive control) using Prism 5 for Mac (GraphPad). FIG. 7 summarizesthe results of this assay. FIG. 7 shows a comparison of compounds Athrough R against HIPPO in their ability to attenuate proliferation ofRAW 264.7 cancer cells. Compounds A through F exhibit similar activityto HIPPO, demonstrating that modification of the substituent of theamide nitrogen outside the tricyclic core is well tolerated andrepresents an auxophore.

1. A compound of formula (I)

wherein Cyc is a 5- to 8-membered monocyclic heterocyclyl ringcomprising one nitrogen atom, with a group of formula Ar¹—CR₂-beingbonded to the ring nitrogen atom; Ar¹ and Ar² are each independentlyaryl groups which are substituted with 0, 1, or 2 J groups; R isindependently H or (C1-C6)alkyl; J is independently (C1-C6)alkyl,(C3-C9)cycloalkyl, (C3-C9)cycloalkyl(C1-C6)alkyl, halo, or(C1-C6)haloalkyl; or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1, wherein the compound is within the subgenus formula(IA)

or a pharmaceutically acceptable salt thereof.
 3. The compound of claim2, wherein Ar¹ and Ar² are each a phenyl group substituted with 0, 1, or2 J groups; and, R at each occurrence is independently H or(C1-C6)alkyl; or a pharmaceutically acceptable salt thereof.
 4. Thecompound of claim 1, wherein the compound is selected from the group

or a pharmaceutically acceptable salt thereof.
 5. A method for treatingcancer, comprising administering an effective amount of a compound offormula (I)

wherein Cyc is a 5- to 8-membered monocyclic heterocyclyl ringcomprising one nitrogen atom, with a group of formula Ar¹—CR₂-beingbonded to the nitrogen atom; Ar¹ and Ar² are aryl groups which aresubstituted with 0, 1, or 2 J groups; R is independently H or(C1-C6)alkyl; J is independently (C1-C6)alkyl, (C3-C9)cycloalkyl,(C3-C9)cycloalkyl(C1-C6)alkyl, halo, or (C1-C6)haloalkyl; or apharmaceutically acceptable salt thereof.
 6. The method of claim 5,wherein the compound is a compound of formula (IA)

or a pharmaceutically acceptable salt thereof.
 7. The method of claim 5,the compound of formula (IA), Ar¹ and Ar² is a phenyl group substitutedwith 0, 1, or 2 J groups.
 8. The method of claim 5, wherein the compoundof formula (I) is formula 2


9. The method of claim 5, wherein the cancer is selected from the groupconsisting of ovarian, colon, breast, liver, pancreas,gastro-intestinal, head- and neck, cervix, prostate, lung cancers,melanomas, glioblastomas, myelomas, neuroblastic-derived CNS tumors,monocytic leukemias, B-cell derived leukemias, T-cell derived leukemias,B-cell derived lymphomas, T-cell derived lymphomas, and mast cellderived tumors, and combinations thereof.